In situ, back-focal-plane-based determination of the numerical apertures in optical microscopes.

In this contribution, we present a technique for in situ determination of the numerical apertures (NAs) of optical microscopes using calibrated diffraction gratings. Many commonly practiced procedures use an external setup to determine the objective and condenser NAs. However, these values may become modified in the used microscope systems, e.g., by system intrinsic apertures. Therefore, in our improved technique, determination of the imaging NA is conducted in situ within the corresponding microscope at hand. Furthermore, the method has been extended to yield the microscope's illumination NA as well. In total, we tested this procedure for determination of the imaging NA for four different microscope objectives with nominal values of 0.55 and 0.9, together with the illumination NAs for four different circular aperture diaphragms with diameters between 10 µm and 500 µm using several gratings of different pitches. All determined NA values agree essentially with their nominal values within their experimental uncertainties, but the uncertainties have been reduced by typically an order of magnitude as compared with the manufacturer's specifications.

Nanomechanical Characterization of Vertical Nanopillars Using an MEMS-SPM Nano-Bending Testing Platform.

Nanomechanical characterization of vertically aligned micro- and nanopillars plays an important role in quality control of pillar-based sensors and devices. A microelectromechanical system based scanning probe microscope (MEMS-SPM) has been developed for quantitative measurement of the bending stiffness of micro- and nanopillars with high aspect ratios. The MEMS-SPM exhibits large in-plane displacement with subnanometric resolution and medium probing force beyond 100 micro-Newtons. A proof-of-principle experimental setup using an MEMS-SPM prototype has been built to experimentally determine the in-plane bending stiffness of silicon nanopillars with an aspect ratio higher than 10. Comparison between the experimental results and the analytical and FEM evaluation has been demonstrated. Measurement uncertainty analysis indicates that this nano-bending system is able to determine the pillar bending stiffness with an uncertainty better than 5%, provided that the pillars' stiffness is close to the suspending stiffness of the MEMS-SPM. The MEMS-SPM measurement setup is capable of on-chip quantitative nanomechanical characterization of pillar-like nano-objects fabricated out of different materials.

Metrological Protocols for Reaching Reliable and SI-Traceable Size Results for Multi-Modal and Complexly Shaped Reference Nanoparticles.

The study described in this paper was conducted in the framework of the European nPSize project (EMPIR program) with the main objective of proposing new reference certified nanomaterials for the market in order to improve the reliability and traceability of nanoparticle size measurements. For this purpose, bimodal populations as well as complexly shaped nanoparticles (bipyramids, cubes, and rods) were synthesized. An inter-laboratory comparison was organized for comparing the size measurements of the selected nanoparticle samples performed with electron microscopy (TEM, SEM, and TSEM), scanning probe microscopy (AFM), or small-angle X-ray scattering (SAXS). The results demonstrate good consistency of the measured size by the different techniques in cases where special care was taken for sample preparation, instrument calibration, and the clear definition of the measurand. For each characterization method, the calibration process is described and a semi-quantitative table grouping the main error sources is proposed for estimating the uncertainties associated with the measurements. Regarding microscopy-based techniques applied to complexly shaped nanoparticles, data dispersion can be observed when the size measurements are affected by the orientation of the nanoparticles on the substrate. For the most complex materials, hybrid approaches combining several complementary techniques were tested, with the outcome being that the reliability of the size results was improved.

A new type of nanoscale reference grating manufactured by combined laser-focused atomic deposition and x-ray interference lithography and its use for calibrating a scanning electron microscope.

Calibration of magnification and nonlinearity of scanning electron microscopy (SEM) is an essential task. In this paper, we proposed a new type of 1D grating sample fabricated by combining laser-focused atomic deposition and x-ray interference lithography as a lateral standard for calibrating SEMs. The calibrations of the grating pattern by a metrological large-range atomic force microscope indicate that the grating sample exhibits outstanding pattern uniformity that surpasses conventional samples fabricated by e-beam lithography: (1) the nonlinear deviation of the grating structures is below +/- 0.5 nm over a measurement range of 5 µm; (2) the maximal variation of the calibrated mean pitch values is lower than 0.01 nm at different locations randomly selected all over the pattern area. The proposed new sample is applied for accurately calibrating the magnification and nonlinearity of a commercial SEM, showing its advantages of easy-of-use and high accuracy. The influence of the defocus level of SEM on the calibration result is also demonstrated. This research offers a feasible solution for highly accurate SEM calibration needed for 3D nanometrology and hybrid metrology demanded in metrology of modern nanoelectronics devices and systems.

Tip wear and tip breakage in high-speed atomic force microscopes.

Tip abrasion is a critical issue particularly for high-speed atomic force microscopy (AFM). In this paper, a quantitative investigation on the tip abrasion of diamond-like-carbon (DLC) coated tips in a high-speed metrological large range AFM device has been detailed. Wear tests are conducted on four different surfaces made of silicon, niobium, aluminum and steel. During the tests, different scanning speeds up to 1 mm/s and different vertical load forces up to approximately 33.2 nN are applied. Various tip characterization techniques such as scanning electron microscopy (SEM) and AFM tip characterizers have been jointly applied to measure the tip form change precisely. The experimental results show that tip form changes abruptly rather than progressively, particularly when structures with steep sidewalls were measured. This result indicates the increased tip breakage risk in high-speed AFM measurements. To understand the mechanism of tip breakage, tip-sample interaction is modelled, simulated and experimentally verified. The results indicate that the tip-sample interaction force increases dramatically in measurement scenarios of steep surfaces.

Using DNA origami nanorulers as traceable distance measurement standards and nanoscopic benchmark structures.

In recent years, DNA origami nanorulers for superresolution (SR) fluorescence microscopy have been developed from fundamental proof-of-principle experiments to commercially available test structures. The self-assembled nanostructures allow placing a defined number of fluorescent dye molecules in defined geometries in the nanometer range. Besides the unprecedented control over matter on the nanoscale, robust DNA origami nanorulers are reproducibly obtained in high yields. The distances between their fluorescent marks can be easily analysed yielding intermark distance histograms from many identical structures. Thus, DNA origami nanorulers have become excellent reference and training structures for superresolution microscopy. In this work, we go one step further and develop a calibration process for the measured distances between the fluorescent marks on DNA origami nanorulers. The superresolution technique DNA-PAINT is used to achieve nanometrological traceability of nanoruler distances following the guide to the expression of uncertainty in measurement (GUM). We further show two examples how these nanorulers are used to evaluate the performance of TIRF microscopes that are capable of single-molecule localization microscopy (SMLM).